Hepatitis B is a major public health problem with more than 350 million persons chronically infected worldwide, 20 to 40% of them being at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Despite the existence of effective preventive vaccines, the hepatitis B virus (HBV) infection is still rampant in many countries, even developed ones, with an estimation of 4.5 millions of new cases of infection per year worldwide. Unlike the WHO recommendation which is to implement universal vaccination, the coverage of full course preventive vaccination varies from 25% in Asia to 75-90% in Europe. Currently hepatitis B is the 10th cause of mortality (around 1 million of deaths/year) and HBV related liver carcinoma, the 5th most frequent cancer. Geographic repartition of HBV infection is uneven with prevalence lower than 1% in Western countries to more than 10% in South Eastern countries, most part of Africa and Equatorial South America. In high HBV chronic carrier prevalence area, vertical transmission from infected mother to neonate is the most frequent mode of contamination and almost always results in chronic hepatitis (90% of cases). This rate can be lowered to 15% by preventive vaccination of infected babies immediately after birth. In Western countries, infection occurs most likely during adulthood through horizontal transmission, via body fluids such as blood, semen, saliva, resulting in acute and self recovering infection in 85% of patients but nevertheless to chronic infection in 15% of cases.
Hepatitis B virus (HBV) is a member of the hepadnaviridae and primarily infects the liver, replicating in hepatocytes. The infectious particles are the so called 42-45 nm “Dane particles” which consist of an outer lipoprotein envelope which contains three different surface proteins (HBs) and an inner nucleocapsid, the major structural protein of which is the core protein (HBcAg). Within the nucleocapsid is a single copy of the HBV genome linked to the viral polymerase protein (P). In addition to 42-45-nm virions, the blood of HBV-infected patients contains 20-nm spheres made of HBcAg and host-derived lipids which are released from infected cells. These spheres outnumber the virions by a factor of 104-106.
The HBV genome is a relaxed circular partially double-stranded DNA of approximately 3,200 nucleotides consisting of a full-length negative strand and a shorter positive strand. It contains 4 overlapping open reading frames (ORFS), C, S, P and X. The C ORF encodes the core protein (or HBcAg), a 183 amino acid-long protein which constitutes the HBV nucleocapsid and a second protein found in the serum of patients during virus replication known as HBcAg which contains a precore N-terminal extension and a part of HBcAg. The C-terminus of the core protein is very basic and contains 4 Arg-rich domains which are predicted to bind nucleic acids as well as numerous phosphorylation sites (the phosphorylation state of core is associated with conformational changes in the capsid particle as described in Vu and Sommers, 1994, J. Virol. 68:2965). The S ORF encodes three surface proteins all of which have the same C terminus but differ at their N-termini due to the presence of three in-frame ATG start codons that divide the S ORF into three regions, S (226 amino acids), pre-S2 (55 amino acids) and pre-S1 (108 amino acids), respectively. The large-surface antigen protein (L) is produced following translation initiation at the first ATG start codon and comprises 389 amino acid residues (preS1-preS2-S). The middle surface antigen protein (M) results from translation of the S region and the pre-S2 region starting at the second start ATG whereas the small surface antigen protein of 226 amino acids (S, also designated HBsAg) results from translation of the S region initiated at the third start ATG codon. The HBV surface proteins are glycoproteins with carbohydrate side chains (glycans) attached by N-glycosidic linkages. The P ORF encodes the viral polymerase and the X ORF contains a protein known as the X protein, which is thought to be a transcriptional activator.
After virions enter hepatocytes, by an as-yet-unknown receptor, nucleocapsids transport the genomic HBV DNA to the nucleus, where the relaxed circular DNA is converted to covalently closed circular DNA (cccDNA). The cccDNA functions as the template for the transcription of four viral RNAs, which are exported to the cytoplasm and used as mRNAs for translation of the HBV proteins. The longest (pre-genomic) RNA also functions as the template for HBV replication, which occurs in nucleocapsids in the cytoplasm. Some of the HBV DNA and polymerase-containing capsids are then transported back to the nucleus, where they release the newly generated relaxed circular DNA to form additional cccDNA. With a half-life longer than the one of hepatocytes, the cccDNA is responsible for the persistence of HBV. Other capsids are enveloped by budding into the endoplasmic reticulum and secreted after passing through the Golgi complex.
A number of preclinical and clinical studies have emphasized the importance of CD4+ and CD8+ T cell immune responses for effective anti-viral response (Ferrari et al, 1990, J Immul, 145:3442; Penna et al, 1996, J Clin Invest, 98:1185; Penna et al, 1997, Hepatology, 25:1022). That is to say, patients naturally having recovered from hepatitis B mounted multi-specific and sustained responses mediated by T helper (TH) and cytotoxic T (CTL) lymphocytes which are readily detectable in peripheral blood. Upon recognition of viral peptides, CTL acquire the capacity to either cure HBV-infected cells via a non-cytopathic, cytokine mediated inhibition of HBV replication and/or to kill them via perforin-Fas ligand and TNFα-mediated death pathways. Both effector functions have been observed during resolution of acute hepatitis B and this type 1 T-cell (Th1) response persists after clinical recovery. It often coincides with an elevation of serum alanine-aminotransferase (ALT) levels and with appearance of HBcAg specific IgM and IgG. Anti-HBe and anti-HBs antibodies appear later and indicate a favorable outcome of infection. HBsAg-specific antibodies are neutralizing, mediate protective immunity and persist for life after clinical recovery. Chronic HBV infection is, however, only rarely resolved by the immune system. When this occurs, viral clearance is associated with increased CTL activity and increased ALT levels caused by a destruction of infected hepatocytes by the immune system. However, the vast majority of chronically infected patients show weak and temporary CD4 and CD8 T cell immune responses that are antigenically restricted and ineffective to clear viral infection, although individual HBV-specific T-cell clones have been isolated and expanded from liver biopsies. The reason for this alteration of the effector functions of the cellular immune response in chronic hepatitis B is currently not known. However it was shown that functional T cell responses can be partially restored in some patients when the viral load is below a threshold of 106 IU/mL (Webster et al 2004, J. Virol, 78:5707). These data are clearly encouraging and emphasize the need for immunomodulatory strategies capable of inducing an effective T-cell response.
Ideally, treatment of chronic viral hepatitis B should first permit to suppress HBV replication before irreversible liver damage, so as to eliminate the virus, prevent disease progression to cirrhosis or liver cancer and improve patient survival. Conventional treatment of chronic hepatitis B includes pegylated interferon-alpha (IFNa) and nucleoside/nucleotide analogues (NUCs) such as lamivudine, and more recently entecavir, telbivudine, adefovir and tenofovir (EASL Clinical Practice Guidelines: management of chronic hepatitis B, 2009). IFNa is a potent antiviral molecule, whereby inhibiting viral replication, which however, causes serious side effects in merely 25-30% of patients. NUCs act as competitive inhibitors of HBV polymerase aimed to inhibit the reverse transcription of the pre-genomic RNA into the negative DNA strand and then the double stranded viral DNA. They limit the formation of new virions, but are ineffective to eliminate the supercoiled cccDNA hidden in the nucleus of infected hepatocytes which constitutes a source of new progeny viruses. This can explain why NUC efficacy is temporary and viral rebound occurs immediately after cessation of treatment, requiring patients to stay life long under treatment. In addition, long-term efficacy is also limited due to emergence of resistant HBV mutants (more than 24% after one year and approximately 66% after four years of lamivudine treatment as discussed in Leung et al., 2001, Hepatology 33:1527) although newer NUCs (entecavir, telbivudine and tenofovir) showed much fewer occurrences of drug-resistant HBV mutants, while increasing suppression of HBV DNA. Long-term treatment data with these new drugs are, however, limited and this higher efficacy has not been correlated with a significantly higher rate of HBs-seroconversion.
Besides antiviral therapies, efforts are currently made to develop supplemental therapies aiming at improved host's immune responses, specifically those mediated by cytotoxic T and helper T lymphocytes. A large majority of existing immunotherapy approaches have focused on the use of HBV surface protein(s), S preS1 and/or preS2 (Smith et al., 1983, Nature 302:490; Lubeck et al., 1989, Proc. Natl. Acad. Sci. USA 86:6763; Adkins et al., 1998, BioDrugs 10:137; Loirat et al., 2000, J. Immunol. 165:4748; Funuy-Ren et al. 2003, J. Med. Virol. 71:376; Kasaks et al., 2004, J. Gen. Virol 85:2665; Xiangming Li et al., 2005, Intern. Immunol. 17:1293; Mancini-Bourguine et al., 2006, Vaccine 24:4482; Vandepapeliere et al., 2007, Vaccine 25:8585). Encouraging results were obtained at least with respect to the stimulation of immune responses. For example, Mancini-Bourguine et al. (2006, Vaccine 24:4482) reported induction and/or recall T cell responses in HBV chronically infected patients injected with a preS2-S-encoding DNA vaccine, which is a good indication that the immune system is still operational in these patients.
HBcAg was also used as an immunogen (Yi-Ping Xing et al., 2005, World J. Gastro, 11:4583) as well as chimeric HBcAg capsids bearing foreign epitopes on their surface (WO92/11368; WO00/32625; Koletzki et al., 1997, J. Gen. Virol. 78:2049). The most promising location for inserting epitopes from the point of view of immunogenicity seems to be the site of an outer loop predicted to be on the surface of HBcAg in the vinicity of position 80 (Argos et al. 1988, EMBO J. 7:819). Schmid et al. (1992, J. Virol, 66:106) and Borisova et al. (1993, J. Virol. 67:3696) were able to insert preS1 and HBsAg epitopes in this region and reported successful immunization with the chimeric particles.
Multivalent vaccine candidates aimed to simultaneously target multiple HBV antigens have also been investigated. Notably, a polyepitope DNA vaccine encoding a fusion polypeptide of multiple cytotoxic T-lymphocytes (CTL) and helper T-lymphocyte (HTL) epitopes present in envelope, core and polymerase proteins was shown to elicit multiple CTL and HTL, responses in preclinical mouse models (Depla et al., 2008, J. Virol, 82:435). Several vaccine formulations based on a mixture of DNA plasmids encoding HBsAg, HBcAg and HBV polymerase were developed (WO2005/056051; WO2008/020656) and demonstrated specific anti-HBV cellular and humoral responses in transgenic mouse model of Chronic hepatitis B (Chae Young Kim et al., 2008, Exp. Mol. Medicine 40:669). Phase I clinical trials were initiated in South Korea in HBV carriers in combination with lamivudine treatment (Yang et al., 2006, Gene Ther. 13:1110).
Accordingly, there still exists a need for alternative immunotherapeutic approaches for inducing immune responses in a more potent and effective manner, especially cell-mediated immune responses, in an individual in need thereof such as an HBV chronically infected patient. Moreover, there is a need to provide vector-based composition capable of expressing the HBV antigen in a stable and sustained manner.
This technical problem is solved by the provision of the embodiments as defined in the claims.
Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.